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In vitro germination to overcome dormancy in seeds of ‘Red Globe’, ‘Italia’ and ‘Niagara Rosada’ grapes

Germinação in vitro para a superação da dormência de sementes de videira ‘Red Globe’, ‘Italia’ e ‘Niagara Rosada’

Abstract

Dormancy in grape seeds slows the progress of many breeding programs of the crop, culminating in low uniformity and low germination percentages. Conventional methods used to overcome dormancy are time-consuming. Thus, in vitro seed germination emerges as a promising alternative to ensure the germination of grape seeds. In this study, we examined the in vitro germination and vigor of seedlings originating from seeds of ‘Red Globe’, ‘Italia’ and ‘Niagara Rosada’ grapes in growth media supplemented with five concentrations of gibberellic acid (GA3) (0, 1.41, 2.83, 4.24 and 5.66 µmol L-1) for 47 days. The use of GA3 increased seed germination percentage and seedling vigor, in the three varieties. Therefore, for the in vitro germination of seeds of ‘Red Globe’ can be used between 1.41 to 4.24 µmol L-1 of GA3. For ‘Italia’ grapes is indicated 1.41 µmol L-1 from GA3 and for ‘Niagara Rosada’ grape is as between 1.41 to 5.66 µmol L-1 from GA3 can be used.

Index terms
Gibberellic acid; plant tissue culture; grapes

Resumo

A dormência em sementes de videira dificulta o avanço de muitos programas de melhoramento genético da cultura, ocasionando desuniformidade e baixa porcentagem de germinação. Os métodos convencionais utilizados para superar a dormência são demorados. Assim, a germinação in vitro das sementes torna-se uma alternativa promissora para garantir a germinação das sementes de videira. Buscou-se avaliar a germinação in vitro e o vigor das plântulas, oriundas de sementes das cultivares de videira ‘Red Globe’, ‘Italia’ e ‘Niagara Rosada’ em meios de cultivo suplementados com cinco concentrações de ácido giberélico (GA3) (0; 1.41; 2.83; 4.24 e 5.66 µmol L-1), por 47 dias. O uso do GA3 promoveu aumento na porcentagem de germinação das sementes e no vigor das plântulas das três variedades. Portanto, para a germinação in vitro de sementes de variedades comerciais de ‘Red Globe’, pode ser utilizado entre 1,41 e 4,24 µmol L-1 de GA3. Para as uvas ‘Italia’, é indicado 1,41 µmol L-1 de GA3, e para a uva ‘Niagara Rosada’ pode ser usado entre 1,41 e 5,66 µmol L-1 de GA3.

Termos para indexação
Ácido giberélico; cultura de tecidos vegetais; uvas

Introduction

In grape breeding programs, seeds obtained from controlled crosses are very important materials of propagation (GISBERT et al., 2018 GISBERT, C.; PEIRÓ, R.; PEDRO, T.S.; OLMOS, A.; JIMÉNEZ, C.; GARCÍA, J. Recovering ancient Grapevine varieties: from genetic variability to in vitro conservation, a case study. In: JORDÃO, A. M.; COSME, F. Grapes and wines: advances in production, processing, analysis and valorization. London: Intech, 2018. p.3-21. Disponível em: https://www.intechopen.com/books/grapes-and-wines-advances-in-production-processing-analysis-and-valorization/recovering-ancient-grapevine-varieties-from-genetic-variability-to-in-vitro-conservation-a-case-stud . Acesso em: 16 abr. 2019. ). However, the low germination percentage of grape seeds constitutes a major obstacle to breeders (WANG et al., 2009 WANG, W.Q.; SONG, S.Q.; LI, S.H.; GAN, Y.Y.; WU, J.H.; CHENG, H.Y. Quantitative description of the effect of stratification on dormancy release of grape seeds in response to various temperatures and water contents. Journal of Experimental Botany, Oxford, v.60, n.12, p.3397–3406, 2009. ; VAL et al., 2010 VAL, A.D.B.; MOTOIKE, S.Y.; ALVARENGA, E.M.; CECON, P.R. Quebra de dormência de sementes da videira cv. niágara rosada se estratificação. Revista Ceres, Viçosa, MG, v.57, n.2, p.234-238, 2010. ). In grape seeds, dormancy is the main cause of low germination percentages (CELIK, 2001 CELIK, H. Effect of bottom heating, germination medium and gibberelic acid treatments on germination of Isabella (Vitislabrusca L.) grapes seeds. Pakistan Journal of Biological Sciences, Faisalabad, v.4, p.953-957, 2001. ; WANG et al., 2009 WANG, W.Q.; SONG, S.Q.; LI, S.H.; GAN, Y.Y.; WU, J.H.; CHENG, H.Y. Quantitative description of the effect of stratification on dormancy release of grape seeds in response to various temperatures and water contents. Journal of Experimental Botany, Oxford, v.60, n.12, p.3397–3406, 2009. ). In the search for methods to overcome dormancy in grape seeds, several authors have suggested cold stratification (BRASIL, 2009 BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes. Brasília (DF): SNDA/DNDV/CLAV, 2009. 365p. ; WANG et al., 2009 WANG, W.Q.; SONG, S.Q.; LI, S.H.; GAN, Y.Y.; WU, J.H.; CHENG, H.Y. Quantitative description of the effect of stratification on dormancy release of grape seeds in response to various temperatures and water contents. Journal of Experimental Botany, Oxford, v.60, n.12, p.3397–3406, 2009. ) along with exogenous application of gibberellic acid (GA3) (ELLIS et al., 1983 ELLIS, R.H.; HONG, T.D.; ROBERTS, E.H. A note on the development of a pratical procedure for promoting the germination of dormant seeds of grape (Vitts spp.). Vitis, Geneva, v.22, p.211-219, 1983. ; ERGENOGLU et al., 1997 ERGENOGLU, F.; TANGOLAR, S.G.K.S. The effects of some pre-treatments for promoting germination of grape seeds. Acta Horticulturae, The Hague, v.44, p.207-212, 1997. ; CELIK, 2001 CELIK, H. Effect of bottom heating, germination medium and gibberelic acid treatments on germination of Isabella (Vitislabrusca L.) grapes seeds. Pakistan Journal of Biological Sciences, Faisalabad, v.4, p.953-957, 2001. ). However, in addition to not being effective in inducing germination in many seeds, these treatments require more than 120 days to produce normal seedlings.

In this scenario, the use of plant tissue culture with in vitro germination of grape seeds is a viable alternative to break dormancy, ensure seed germination and provide new genotypes for grape breeding programs. The present study proposes to examine in vitro germination and vigor of seedlings originating from seeds of table grapes in growth media supplemented with five concentrations of GA3 (0, 1.41, 2.83, 4.24 and 5.66 μmol L-1).

The plant material consisted of seeds of three commercial genotypes of table grape, namely, ‘Red Globe’, ‘Italia’ and ‘Niagara Rosada’. Seeds were removed from the pericarp, washed in running water, disinfected with neutral detergent, left to dry at room temperature for 48 h and then stored in a refrigerator (±4°C) for seven days, until the in vitro germination test was assembled.

The experiment was set up as a completely randomized design with a 3 × 5 factorial arrangement in which the in vitro germination of three table grape genotypes was tested in growth media with five concentrations of GA3 (0, 1.41, 2.83, 4.24 and 5.66 μmol L-1). Seven replicates were used, each of which was composed of a culture bottle containing 40 mL of growth medium and five seeds.

Seeds were cut in the micropyle region and in the upper region using cuticle pliers, in accordance with the methodology of Val et al. (2010) VAL, A.D.B.; MOTOIKE, S.Y.; ALVARENGA, E.M.; CECON, P.R. Quebra de dormência de sementes da videira cv. niágara rosada se estratificação. Revista Ceres, Viçosa, MG, v.57, n.2, p.234-238, 2010. . In a laminar flow hood, the seeds were then disinfected in 70% alcohol for 30 s, then in for 20 min a 1.0% sodium hypochlorite (NaClO) solution with two drops of Tween® 20 deposited for every 100 mL and lastly washed three times in autoclaved deionized water. The seeds were inoculated in growth medium constituted by half of the concentrations of MS medium mineral salts (MURASHIGE and SKOOG, 1962 MURASHIGE, T.; SKOOG, F.A. Revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum, Copenhagen, v.15, p.437-497, 1962. ), White vitamins, 100 mg L-1 myo-inositol, 30 g L-1 sucrose, 200 mg L-1 polyvinylpyrrolidone (PVP) and five concentrations of GA3 (Vetec®) (0, 1.41, 2.83, 4.24 and 5.66 μmol L-1), with the pH adjusted to 5.7, and solidified with 7 g L-1 pure bacteriological agar (Vetec®). The growth medium was autoclaved for 20 min at 121 °C and 1.1 atm and 40 mL were distributed per culture bottle. The growth regulator GA3 was added to the growth medium after autoclaving.

The bottles with the seeds were kept in the growth room at a temperature of 27±2 °C, photoperiod of 16:8 (light:dark) and luminosity supplied by Osram® daylight lamps with a luminous intensity of 54 μmol m-2 s-1. After 47 days of growth, the seeds were examined for the germination percentage of normal seedlings, abnormal seedlings, seeds with primary root emission and ungerminated seeds (seedlings were considered normal only when showing expanded shoots and roots) and the vigor variables of number of leaves, shoot length, root length, total dry matter and germination speed index (GSI), which was evaluated at a two-day interval, during the 47 days of germination, by observing the number of seeds producing primary roots (MAGUIRE, 1962 MAGUIRE, J.D. Speed of germination and in selection and evaluation for seedling emergence and vigor. Crop Science, Madison, v.2, n.2, p.176-177, 1962. ).

The variables were subjected to an initial normality test (Shapiro-Wilk). Subsequently, an analysis of variance was performed and means were separated by the t test (LSD) at P<0.05, using Sisvar statistical software (FERREIRA, 2011 FERREIRA, D.F. Sisvar: a computer statistical analysis system. Ciência e Agrotecnologia, Lavras, v.5, n.6, p.1039-1042, 2011. ).

Dormancy is characterized by a condition in which some seeds do not germinate even when exposed to favorable environmental conditions (BASKIN and BASKIN, 2004 BASKIN, C.C.; BASKIN, J.M. A classification system for seed dormancy. Seed Science Research, Cambridge, v.14, p.1-16, 2004. ). In grape seeds, one of the reported types of dormancy is physical, caused by the thickness and hardness of the seed integument, which preclude the entry of water into the embryo (ELLIS et al., 1983 ELLIS, R.H.; HONG, T.D.; ROBERTS, E.H. A note on the development of a pratical procedure for promoting the germination of dormant seeds of grape (Vitts spp.). Vitis, Geneva, v.22, p.211-219, 1983. ). In in vitro conditions, Val et al. (2010) VAL, A.D.B.; MOTOIKE, S.Y.; ALVARENGA, E.M.; CECON, P.R. Quebra de dormência de sementes da videira cv. niágara rosada se estratificação. Revista Ceres, Viçosa, MG, v.57, n.2, p.234-238, 2010. suggested making cuts on the seed integument to facilitate water imbibing by the embryo. Thus, the cuts made in the micropyle region and in the upper region, in our study, managed to overcome physical dormancy and hydrate the embryo.

However, differences were observed for the germination and percentage results across the studied varieties. ‘Itlia’ grape exhibited the highest percentage of germination into normal seedlings (58.5%), differing statistically from ‘Red Globe’ (43.9%) and ‘Niagara Rosada’ (53.6%) (Figure 1a). At 47 days of germination in vitro, several ‘Italia’ seeds (16.6%) inoculated in growth medium with 1.41 μmol L-1 GA3 were still producing primary root (Table 1).

Figure 1
In vitro germination means of normal seedlings of three grape varieties (a) in culture medium containing five concentrations of GA3 (b) after 47 days of cultivation. In vitro germination means of abnormal seedlings of three grape varieties (c) in culture medium containing five concentrations of GA3 (d) after 47 days of cultivation. Roots length of three grape varieties seedling (e) placed to germinate in vitro in medium containing five concentrations of GA3 (f) for 47 days. Means followed by the same lowercase letter do not differ by t test (LSD) at p <0.05.

Table 1
The interaction between three grape seeds varieties and five concentrations of GA3 after 47 days of in vitro germination, for the variables: seeds with primary root emission and ungerminated seeds.

Additionally, the highest means observed for the vigorrelated variables were obtained by varieties ‘Red Globe’ (number of leaves, shoot length and dry matter) and ‘Italia’ (GSI and number of leaves) (Table 2). For root length, however, no difference was detected between the three varieties (Figure 1e).

Table 2
The interaction between three grape seeds varieties and five concentrations of GA3 after 47 days of in vitro germination, for the variables: GSI, number of leaves, shoot length and dry matter.

The use of GA3 as a plant growth regulator provided a significant increase in seed germination percentage across the grape varieties, which rose from 13% (treatment without GA3) to more than 56%, irrespective of the concentration (Figure 1b). Lack of GA3 in the growth medium resulted in the highest percentages of ungerminated seeds for all studied varieties (Table 1). The use of GA3 provided an increase in seedling vigor through GSI for ‘Italia’ grape and in shoot length for varieties Rev. Bras. Frutic., Jaboticabal, 2019, v. 41, n. 5: (e-495) ‘Red Globe’ and ‘Italia’. Additionally, GA3 considerably increased germination rate and speed in the three grape varieties (Figure 2). However, addition of 2.83 μmol L-1 of GA3 to the growth medium led to the emergence of abnormal seedlings (10.4%) (Figure 1d).

Figure 2
In vitro germination speed of the grape varieties Red Globe, Italia and Niagara Rosada during a period of 47 days of in vitro germination in culture medium containing five concentrations of GA3 (without, 1,41, 2,83, 4,24 e 5,66 µmol L-1).

A positive effect of GA3 on the in vitro germination percentage of ‘Niagara Rosada’ seeds was reported by Val et al. (2010) VAL, A.D.B.; MOTOIKE, S.Y.; ALVARENGA, E.M.; CECON, P.R. Quebra de dormência de sementes da videira cv. niágara rosada se estratificação. Revista Ceres, Viçosa, MG, v.57, n.2, p.234-238, 2010. and in ‘Isabella’ grape by Celik (2001) CELIK, H. Effect of bottom heating, germination medium and gibberelic acid treatments on germination of Isabella (Vitislabrusca L.) grapes seeds. Pakistan Journal of Biological Sciences, Faisalabad, v.4, p.953-957, 2001. , in different substrates. In seeds, GA3 acts on the synthesis and activation of the hydrolytic enzymes present in the endosperm tissue (MOSHKOV et al., 2008 MOSHKOV, I.E.; NOVIKOVA, G.V.; HALL, M.A.; GEORGE, E.F. Plant Growth Regulators III: Gibberellins, Ethylene, Abscisic Acid, their Analogues and Inhibitors; Miscellaneous Compounds. In: GEORGE, E.F.; HALL, M.A.; KLERT, G.D. Plant propagation by tissue culture. 3rd ed. Dordrecht: Springer, 2008. p.227-282. ).

The conventional method provided by the Rules for Seed Testing (RST) for dormancy breaking in seeds of Vitis vulpina indicates pre-chilling in wet substrate at a temperature of 3-5ºC for a period of three months, to only then set them to germinate (BRASIL, 2009 BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes. Brasília (DF): SNDA/DNDV/CLAV, 2009. 365p. ). The entire process may exceed 120 days. By contrast, in vitro germination of grape seeds with GA3 supplementation is a promising strategy in grape breeding programs, as it allows for breaking seed dormancy, accelerating the germination period and obtaining vigorous seedlings (Figure 3) in 47 days. The significant variability observed among the three varieties was due to the genotypic difference between species. Therefore, for the in vitro germination of seeds of ‘Red Globe’ can be used between 1.41 to 4.24 μmol L-1 of GA3. For ‘Italia’ grapes is indicated 1.41 μmol L-1 from GA3 and for ‘Niagara Rosada’ grape is as between 1.41 to 5.66 μmol L-1 from GA3 can be used.

Figure 3
Normal seedlings of Red Globe grape after 47 days of in vitro germination in mediun culture: without GA3 (a), with 1.41 µmol L-1 of GA3 (b), 2.83 µmol L-1 of GA3 (c), 4.24 µmol L-1 of GA3 (d) and 5.66 µmol L-1 of GA3 (e).

Acknowledgements

The authors thank the Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF) and the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), for the financial support during this research and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

  • BASKIN, C.C.; BASKIN, J.M. A classification system for seed dormancy. Seed Science Research, Cambridge, v.14, p.1-16, 2004.
  • BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes. Brasília (DF): SNDA/DNDV/CLAV, 2009. 365p.
  • CELIK, H. Effect of bottom heating, germination medium and gibberelic acid treatments on germination of Isabella (Vitislabrusca L.) grapes seeds. Pakistan Journal of Biological Sciences, Faisalabad, v.4, p.953-957, 2001.
  • ELLIS, R.H.; HONG, T.D.; ROBERTS, E.H. A note on the development of a pratical procedure for promoting the germination of dormant seeds of grape (Vitts spp.). Vitis, Geneva, v.22, p.211-219, 1983.
  • ERGENOGLU, F.; TANGOLAR, S.G.K.S. The effects of some pre-treatments for promoting germination of grape seeds. Acta Horticulturae, The Hague, v.44, p.207-212, 1997.
  • FERREIRA, D.F. Sisvar: a computer statistical analysis system. Ciência e Agrotecnologia, Lavras, v.5, n.6, p.1039-1042, 2011.
  • GISBERT, C.; PEIRÓ, R.; PEDRO, T.S.; OLMOS, A.; JIMÉNEZ, C.; GARCÍA, J. Recovering ancient Grapevine varieties: from genetic variability to in vitro conservation, a case study. In: JORDÃO, A. M.; COSME, F. Grapes and wines: advances in production, processing, analysis and valorization. London: Intech, 2018. p.3-21. Disponível em: https://www.intechopen.com/books/grapes-and-wines-advances-in-production-processing-analysis-and-valorization/recovering-ancient-grapevine-varieties-from-genetic-variability-to-in-vitro-conservation-a-case-stud . Acesso em: 16 abr. 2019.
  • MAGUIRE, J.D. Speed of germination and in selection and evaluation for seedling emergence and vigor. Crop Science, Madison, v.2, n.2, p.176-177, 1962.
  • MOSHKOV, I.E.; NOVIKOVA, G.V.; HALL, M.A.; GEORGE, E.F. Plant Growth Regulators III: Gibberellins, Ethylene, Abscisic Acid, their Analogues and Inhibitors; Miscellaneous Compounds. In: GEORGE, E.F.; HALL, M.A.; KLERT, G.D. Plant propagation by tissue culture. 3rd ed. Dordrecht: Springer, 2008. p.227-282.
  • MURASHIGE, T.; SKOOG, F.A. Revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum, Copenhagen, v.15, p.437-497, 1962.
  • VAL, A.D.B.; MOTOIKE, S.Y.; ALVARENGA, E.M.; CECON, P.R. Quebra de dormência de sementes da videira cv. niágara rosada se estratificação. Revista Ceres, Viçosa, MG, v.57, n.2, p.234-238, 2010.
  • WANG, W.Q.; SONG, S.Q.; LI, S.H.; GAN, Y.Y.; WU, J.H.; CHENG, H.Y. Quantitative description of the effect of stratification on dormancy release of grape seeds in response to various temperatures and water contents. Journal of Experimental Botany, Oxford, v.60, n.12, p.3397–3406, 2009.

Publication Dates

  • Publication in this collection
    14 Nov 2019
  • Date of issue
    2019

History

  • Received
    02 May 2019
  • Accepted
    22 Aug 2019
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